Hepatocytes are parenchymal liver cells, and make up 60-80% of the cytoplasmic mass of the liver. Hepatocytes play a key role in the detoxification, modification and excretion of exogenous and endogenous substances (Ponsoda, X. et al. (2004) “Drug Metabolism By Cultured Human Hepatocytes: How Far Are We From The In Vivo Reality?” Altern Lab Anim. 32(2): 101-110). One of the detoxifying functions of hepatocytes is to modify ammonia to urea for excretion. They are also important in protein synthesis and storage, in the transformation of carbohydrates and in the synthesis of cholesterol, bile salts and phospholipids (Postic, C. et al. (2004) “Role Of The Liver In The Control Of Carbohydrate And Lipid Homeostasis,” Diabetes Metab. 30(5):398-408). The hepatocyte is the only cell in the body that manufactures albumin, fibrinogen, and the prothrombin group of clotting factors. It is the main site for the synthesis of lipoproteins, ceruloplasmin, transferrin, and glycoproteins. Hepatocytes manufactures their own structural proteins and intracellular enzymes. Hepatocytes are also important depots for vitamin B12 and iron.
Due to these attributes, isolated and cultured hepatocytes have become very attractive as models systems for the study of liver functions (Chesne, C. et al. (1993) “Viability And Function In Primary Culture Of Adult Hepatocytes From Various Animal Species And Human Beings After Cryopreservation,” Hepatology 18(2):406-414; Guillouzo, A. et al. (1986) “Isolated and Cultured Hepatocytes,” Paris: les Editions INSERM and London: John Libbey Eurotext); Ponsoda, X. et al. (2004) “Drug Metabolism By Cultured Human Hepatocytes: How Far Are We From The In Vivo Reality?” Altern Lab Anim. 32(2): 101-110; Gomez-Lechon, M. J. et. al. (2004) “Human Hepatocytes In Primary Culture: The Choice To Investigate Drug Metabolism In Man,” Curr Drug Metab. 5(5):443-462; Lemaigre, F. et al. (2004) “Liver Development Update: New Embryo Models, Cell Lineage Control, And Morphogenesis,” Curr Opin Genet Dev. 14(5):582-590; Nanji, A. A. (2004) “Animal Models Of Nonalcoholic Fatty Liver Disease And Steatohepatitis,” Clin Liver Dis. 8(3):559-574; Hewitt, N. J. et al. (2004) Cryopreserved Rat, Dog And Monkey Hepatocytes: Measurement Of Drug Metabolizing Enzymes In Suspensions And Cultures,” Hum Exp Toxicol. 23(6):307-316).
In addition to their use in liver models, hepatocytes have the potential of being used to produce Bioartificial Livers (BALs) or in hepatocyte transplantation that can provide liver functions for individuals suffering from liver disease or liver failure. Bioartificial Livers (BALs) are described by Anand, A. C. (1996) “Bioartificial Livers: The State Of The Art,” Trop Gastroenterol. 17(4):197-198, 202-211; Gan, J. H. et al. (2005) “Hybrid Artificial Liver Support System For Treatment Of Severe Liver Failure,” World J Gastroenterol. 11 (6):890-894; Fukuda, J. et al. (2004) “Hepatocyte Organoid Culture In Elliptic Hollow Fibers To Develop A Hybrid Artificial Liver,” Int J Artif Organs. 27(12): 1091-1099; Meng, Q. et al. (2004) “Hepatocyte Culture In Bioartificial Livers With Different Membrane Characteristics,” Biotechnol Lett. 26(18):1407-1412; Sekido, H. et al. (2004) “Usefulness Of Artificial Liver Support For Pretransplant Patients With Fulminant Hepatic Failure,” Transplant Proc. 36(8):2355-2356; WO03/105663A2, WO05/000376A2, and U.S. Pat. No. 6,759,245. Hepatocyte transplantation is described by Chan, C. et al. (2004) “Hepatic Tissue Engineering For Adjunct And Temporary Liver Support: Critical Technologies,” Liver Transpl. 10(11): 1331-1342; Lee, S. W. et al. (2004) “Hepatocyte Transplantation: State Of The Art And Strategies For Overcoming Existing Hurdles,” Ann. Hepatol. 3(2):48-53; Horslen, S. P. (2004) “Hepatocyte Transplantation,” Transplantation 77(10):1481-1486; Burlina, A. B. (2004) “Hepatocyte Transplantation For Inborn Errors Of Metabolism,” J. Inherit. Metab. Dis. 27(3):373-83; and Fox, I. J. et al. (2004) “Hepatocyte Transplantation,” Am. J. Transplant. 4 Suppl. 6:7-13.
A limiting factor in the development of such model systems and to the development of Bioartificial Livers (BALs) has been the erratic source and limited availability of hepatocytes, especially human hepatocytes. Fresh hepatocytes are obtainable only from liver resections or non-transplantable livers of multi-organ donors (Lloyd, T. D. R. et al. (2003) Cryopreservation Of Hepatocytes: A Review Of Current Methods For Banking,” Cell and Tissue Culture Banking 4:3-15). The supply of such tissue is inconsistent and often geographically inconvenient in light of the limited functional lifespan of liver tissue (Smrzova, J. et al. (2001) “Optimization Of Porcine Hepatocytes Cryopreservation By Comparison Of Viability And Enzymatic Activity Of Fresh And Cryopreserved Cells,” Acta Veterinaria Brunensis 70:141-147).
One approach to addressing this problem has involved the development of hepatocyte storage conditions that allow hepatocytes to be maintained over time with their cellular functions preserved. Cryopreservation methods for the storage of hepatocytes have been developed to address this need (see, Lloyd, T. D. R. et al. (2003) Cryopreservation Of Hepatocytes: A Review Of Current Methods For Banking,” Cell and Tissue Culture Banking 4:3-15; Loretz, L. J. et al. (1989) “Optimization Of Cryopreservation Procedures For Rat And Human Hepatocytes,” Xenobiotica. 19(5):489-498; Shaddock, J, G. et al. (1993) “Cryopreservation And Long-Term Storage Of Primary Rat Hepatocytes: Effects On Substrate-Specific Cytochrome P450-Dependent Activities And Unscheduled DNA Synthesis,” Cell Biol Toxicol. 9(4):345-357; Novicki, D. L. et al. (1982) “Cryopreservation Of Isolated Rat Hepatocytes,” In Vitro. 18(4):393-399; Zaleski, J. et al. (1993) “Preservation Of The Rate And Profile Of Xenobiotic Metabolism In Rat Hepatocytes Stored In Liquid Nitrogen ,” Biochem Pharmacol. 46(1):111-116). Typically, such measures comprise storage in liquid nitrogen (−196° C.) or in frozen nitrogen gas (−150° C.). The ability to recover viable thawed cells has been found to depend on multiple factors such as the rate of freezing, the concentration of hepatocytes, the type of cryoprotectant employed, and the final cooling temperature. Cell concentrations of 106-107 cells/ml have been typically employed. The isolated hepatocytes are typically incubated in suspension for a period (e.g., 4-48 hours) to allow them to recover from the isolation process. Thereafter, a cryoprotectant (such as glycerol, DMSO, polyvinylpyrrolodine, or dextran) is added, and the hepatocytes are frozen. The art has developed various freezing procedures, all designed to minimize or prevent the occurrence of intracellular ice. The freezing rates typically vary from −0.05° C./min to −50° C./min (see, Lloyd, T. D. R. et al. (2003) Cryopreservation Of Hepatocytes: A Review Of Current Methods For Banking,” Cell and Tissue Culture Banking 4:3-15).
While the development of cryopreservation methods for the storage of hepatocytes has significantly facilitated the availability of human hepatocytes, cryopreservation has been found to cause a significant decrease in cellular viability (e.g., 25-35%) (Dou, M. et al. (1992) “Thawed Human Hepatocytes In Primary Culture,” Cryobiology 29:454-469; Alexandre, E. et al. (2002) “Cryopreservation Of Adult Human Hepatocytes Obtained From Resected Liver Biopsies,” Cryobiology 44:103-113). Coundouris, J. A. et al. (1993) reported viability of 67% after 24 hours, declining to 49% after 14 days (Coundouris, J.A. et al. (1993) “Cryopreservation Of Human Adult Hepatocytes For Use In Drug Metabolism And Toxicity Studies,” Xenobiotica. 23(12):1399-1409). Adams, R. M. et al. have reported that the viability of hepatocytes may be enhanced to greater than 90% using specialized cyropreservation fluids, however, only 16% of cells were found to be capable of replication (Adams, R. M. et al. (1995) “Effective Cryopreservation And Long-Term Storage Of Primary Human Hepatocytes With Recovery Of Viability, Differentiation, And Replicative Potential,” Cell Transplant. 4(6):579-586). Methods of cryopreservation are disclosed in U.S. Pat. Nos. 5,795,711, 6,136,525, 5,895,745; International Patent Publications WO04/009766, WO92/12722, WO/0153462, European Patent No. EP0834252B, and U.S. Patent Application Publication Nos. US20020039786A1, US20030134418A1. The poor recovery of cells when cryopreserved continues to limit the use of hepatocytes in in vitro liver models.
A second major problem affecting the use of both fresh and cryopreserved hepatocytes is the variation of liver enzyme expression that is observed in tissue from different donors (Li, A. P. et al. (1999) “Present Status Of The Application Of Cryopreserved Hepatocytes In The Evaluation Of Xenobiotics: Consensus Of An International Expert Panel,” Chem Biol Interact. 121(1):117-123; Li, A. P. et al. (1999) “Cryopreserved Human Hepatocytes: Characterization Of Drug-Metabolizing Enzyme Activities And Applications In Higher Throughput Screening Assays For Hepatotoxicity, Metabolic Stability And Drug-Drug Interaction Potential,” Chem Biol Interact. 121(1):17-35; O'Brien, Z. Z. et al. (undated) “The Construction Of A Representative Human Cryopreserved Hepatocyte Pool For Metabolism Study.” One solution to this sample variation involves pooling samples from different sources to produce a “composite” hepatocyte preparation having the characteristics of “average” liver cells. However, the frequency of receipt of fresh tissue and the need to cryopreserve hepatocytes immediately after isolation has been cited as preventing the preparation of hepatocyte pools. Thus, multiple companies (e.g., Xenotech, LLC; BD Biosciences) refrain from selling pooled hepatocytes thus forcing the end user to thaw and pool hepatocytes from several different donors. This difficulty remains even though pooled cryopreserved human hepatocytes are a valid model for metabolic studies (Zhang, J. G. et al. (undated) “Validation Of Pooled Cryopreserved Human Hepatocytes As A Model For Metabolic Studies.”
Thus, despite all prior advances, a need remains for processes that would enable the availability of hepatocytes for medical research and other purposes. A need further exists for a stable and reproducible source of human hepatocytes. The present invention permits the production and availability of hepatocyte preparations that may be repeatedly cryopreserved and thawed without unacceptable loss of viability. The invention thus permits multiple hepatocyte samples to be pooled to produce pooled hepatocyte preparations, especially pooled cryopreserved human hepatocyte preparations. Using such advance, pooled cryopreserved human hepatocytes are now commercially available from In Vitro Technologies (Baltimore, Md.).